Method for treating inflammation associated with amyloid deposits and brain inflammation involving activated microglia

ABSTRACT

Filamentous bacteriophage which does not display an antibody or a non-filamentous bacteriophage antigen on its surface is used to inhibit or treat brain inflammation associated with amyloid deposits and/or involving activated microglia, to inhibit the formation of amyloid deposits, and to disaggregate pre-formed amyloid deposits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/815,294,which is a 371 national stage application of PCT/US06/03291, filed Jan.31, 2006, which claims priority from U.S. provisional application No.60/648,383, filed Feb. 1, 2005, the entire contents of whichapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to inhibiting amyloid deposit formation anddissolving pre-formed amyloid deposits and to methods and pharmaceuticalcompositions for treating brain inflammation and for treatinginflammation associated with amyloid or amyloid-like deposits within thebrain and elsewhere in the body.

2. Description of the Related Art

Plaque forming diseases are characterized by the presence of amyloidplaques deposits in the brain as well as neuronal degeneration. Amyloiddeposits are formed by peptide aggregated into an insoluble mass. Thenature of the peptide varies in different diseases but in most cases,the aggregate has a beta-pleated sheet structure and stains with CongoRed dye. In addition to Alzheimer's disease (AD), which includes earlyonset Alzheimer's disease, late onset Alzheimer's disease, andpresymptomatic Alzheimer's disease, other diseases characterized byamyloid deposits are, for example, SAA amyloidosis, hereditary Icelandicsyndrome, multiple myeloma, and prion diseases. The most common priondiseases in animals are scrapie of sheep and goats and bovine spongiformencephalopathy (BSE) of cattle (Wilesmith and Wells, 1991). Four priondiseases have been identified in humans: (i) kuru, (ii)Creutzfeldt-Jakob Disease (CJD), (iii) Gerstmann-Streussler-SheinkerDisease (GSS), and (iv) fatal familial insomnia (FFI) (Gajdusek, 1977;and Tritschler et al. 1992).

Prion diseases involve conversion of the normal cellular prion protein(PrPC) into the corresponding scrapie isoform (PrPSc). Spectroscopicmeasurements demonstrate that the conversion of PrPC into the scrapieisoform (PrPSc) involves a major conformational transition, implyingthat prion diseases, like other amyloidogenic diseases, are disorders ofprotein conformation. The transition from PrPC to PrPSc is accompaniedby a decrease in α-helical secondary structure (from 42% to 30%) and aremarkable increase in β-sheet content (from 3% to 43%) (Caughey et al,1991; and Pan et al, 1993). This rearrangement is associated withabnormal physiochemical properties, including insolubility innon-denaturing detergents and partial resistance to proteolysis.Previous studies have shown that a synthetic peptide homologous withresidues 106-126 of human PrP (PrP106-126) exhibits some of thepathogenic and physicochemical properties of PrPSc (Selvaggini et al,1993; Tagliavini et al, 1993; and Forloni et al, 1993). The peptideshows a remarkable conformational polymorphism, acquiring differentsecondary structures in various environments (De Gioia et al, 1994). Ittends to adopt a β-sheet conformation in buffered solutions, andaggregates into amyloid fibrils that are partly resistant to digestionwith protease. Recently, the X-ray crystallographic studies of a complexof antibody 3F4 and its peptide epitope (PrP 104-113) provided astructural view of this flexible region that is thought to be acomponent of the conformational rearrangement essential to thedevelopment of prion disease (Kanyo et al, 1999). The identification ofclasses of sequences that participate in folding-unfolding and/orsolubilization-aggregation processes may open new direction for thetreatment of plaque forming disease, based on the prevention ofaggregation and/or the induction of disaggregation (Silen and Agard,1989; Frenkel et al, 1998; Horiuchi and Caughey, 1999).

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. Broadly speaking, the disease falls into two categories: lateonset, which occurs in old age (typically above 65 years) and earlyonset, which develops well before the senile period, e.g., between 35and 60 years. In both types of the disease, the pathology is similar,but the abnormalities tend to be more severe and widespread in casesbeginning at an earlier age. The disease is characterized by two typesof lesions in the brain, senile plaques and neurofibrillary tangles.Senile plaques are areas of disorganized neutrophils up to 150 mm acrosswith extracellular amyloid deposits at the center, visible bymicroscopic analysis of sections of brain tissue. Neurofibrillarytangles are intracellular deposits of tau protein consisting of twofilaments twisted about each other in pairs.

The principal constituent of the senile plaques is a peptide termedamyloid beta (Aβ) or beta-amyloid peptide (βAP or) βA). The amyloid betapeptide is an internal fragment of 39-43 amino acids of a precursorprotein termed amyloid precursor protein (APP). Several mutations withinthe APP protein have been correlated with the presence of Alzheimer'sdisease (Goate et al, (1991), valine717 to isoleucine; Chartier Harlanet al, (1991), valine717 to glycine; Murrell et al, (1991), valine717 tophenylalanine; Mullan et al, (1992), a double mutation, changinglysine595-methionine596 to asparagine595-leucine596).

Such mutations are thought to cause Alzheimer's disease by increased oraltered processing of APP to beta-amyloid, particularly processing ofAPP to increased amounts of the long form of beta-amyloid (i.e., Aβ1-42and Aβ1-43). Mutations in other genes, such as the presenilin genes, PS1and PS2, are thought indirectly to affect processing of APP to generateincreased amounts of long form beta-amyloid (see Hardy, TINS 20, 154,1997). These observations indicate that beta-amyloid, and particularlyits long form, is a causative element in Alzheimer's disease.

Other peptides or proteins with evidence of self aggregation are alsoknown, such as, but not limited to, amylin (Young et al, 1994);bombesin, cerulein, cholecystokinin octapeptide, eledoisin,gastrin-related pentapeptide, gastrin tetrapeptide, somatostatin(reduced), substance P; and peptide, luteinizing hormone releasinghormone, somatostatin N-Tyr (Banks and Kastin, 1992).

Publications on amyloid fibers indicate that cylindrical β-sheets arethe only structures consistent with some of the x-ray and electronmicroscope data, and fibers of Alzheimer Aβ fragments and variants areprobably made of either two or three concentric cylindrical β-sheets(Perutz et al., 2002). The complete Aβ peptide contains 42 residues,just the right number to nucleate a cylindrical shell; this finding andthe many possible strong electrostatic interactions in β-sheets made ofthe Aβ peptide in the absence of prolines account for the propensity ofthe Aβ peptide to form the extracellular amyloid plaques found inAlzheimer patients. If this interpretation is correct, amyloid consistsof narrow tubes (nanotubes) with a central water-filled cavity.Reversibility of amyloid plaque growth in-vitro suggests steady-stateequilibrium between βA in plaques and in solution (Maggio and Mantyh,1996). The dependence of βA polymerization on peptide-peptideinteractions to form a β-pleated sheet fibril, and the stimulatoryinfluence of other proteins on the reaction, suggest that amyloidformation may be subject to modulation. Many attempts have been made tofind substances able to interfere with amyloid formation. Among the mostinvestigated compounds are antibodies, peptide composed of beta-breakeramino acids like proline, addition of charged groups to the recognitionmotif and the use of N-methylated amino-acid as building blocks(reviewed by Gazit, 2002).

Cyclic peptides made of alternate D and L residues form such nanotubesthat kill bacteria by inserting themselves into membranes anddepolarizing them (Perutz et al., 2002). There is some suggestion thatsome amyloid fibers might be conductors and kill cells by the samemechanism.

Aromatic compounds such as congo red that can insert themselves intogaps between helical turns might destabilize the cylindrical shells andinitiate this process, but prevention would be more effective andprobably easier to achieve (Perutz et al., 2002).

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention provides a method for inhibiting or treatinginflammation associated with amyloid deposits within the brain andelsewhere in the body and brain inflammation involving activatedmicroglia. The method involves administering to a patient in needthereof an effective amount of a wild-type filamentous bacteriophage ora filamentous bacteriophage which does not display an antibody or anon-filamentous bacteriophage antigen on its surface.

The present invention also provides a pharmaceutical compositioncontaining an effective amount of the filamentous bacteriophage as theactive ingredient.

The present further provides methods for inhibiting the formation ofamyloid deposits or for disaggregating pre-formed amyloid deposits bycontacting filamentous bacteriophage with plaque forming peptides orpre-formed amyloid deposits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a cross-section of the brainshowing intracranial application of phage/PBS to hAPP transgenic mice.Phages were injected intracerebrally to five transgenic mice. To thecontralateraly hemisphere, PBS was injected. The mice were sacrificedafter 1 hour, 2 days and 3 days. Their brain were cut to 5 μm sections,and are being stained with Thioflavine-S to evaluate their plaque load.

FIGS. 2A and 2B are graphs showing disaggregation (FIG. 2A) andprevention (FIG. 2B) of β-amyloid 1-40 aggregation by filamentous phageusing the ThT assay. In FIG. 2A (disaggregation test), β-amyloid (54 μM)was incubated for 21 days alone. On the 21^(th) day, different phageconcentrations were added and incubated overnight. In FIG. 2B(prevention test) β-amyloid (58 μM) was incubated either alone or withfilamentous phage for 7 days, then the samples were added to ThTsolution and amyloid content was measured by spectrofluorimeter. Amyloidcontent was measured using Thioflavin-T which binds to amyloid fibrilsand its fluorescent emission was detected at 485 nm wavelength.

FIGS. 3A-3K are electron micrographs of β-amyloid which was incubated inthe absence or presence of filamentous phage (magnification ×100K). FIG.3A: β-amyloid 97 μM in PBS in the absence of phage. β-amyloid wasstained with mouse monoclonal antibody 10D5 followed by goat anti mouse12 mn gold conjugated antibody. FIGS. 3B-3C: β-amyloid 97 μM incubatedwith 0.5 nM (1×10¹⁰) phages. β-amyloid was stained with mouse monoclonalantibody 10D5 and 2nd antibody conjugated to 12 mn gold particle. Thephage was labeled with polyclonal rabbit anti-phage sera, followed bygoat anti-rabbit 6 nm gold conjugated antibody. No signal of thosesecond antibodies was observed in samples containing no phage or no1^(st) antibodies. FIG. 3D: Filamentous phage (fd) (1×10¹⁰ phages) wasstained with rabbit polyclonal sera. The 2nd antibody was conjugated to6 nm gold particle. FIG. 3E: Filamentous phage near beta amyloid. Thepeptide probably enhances the phage degradation as the scatteredlabeling with anti-phage antibody shows (arrow). This labeling is absentwhen phage is incubated alone (sample D). FIGS. 3F-3G; β-amyloid aloneafter 10 days of incubation at 37° C. (magnification: F-×30K, G-×100K).FIGS. 3H-3I: β-amyloid was incubated alone for 10 days, on the 10th dayfilamentous phages were added for another 16 hours (H-×30K, I-×100Kenlargement of H). FIGS. 3J-3K: Interface between amyloid fibril andfilamentous phage. The phages are organized along the fibril axis(×100K).

FIGS. 4A-4C are electron micrographs where spherical phages were addedto preaggregated β-amyloid and incubated together overnight. β-amyloidwith spherical phages (FIGS. 4A and 4B). Spherical phage alone (FIG. 4C)(magnification ×100K).

FIGS. 5A-5C are graphs showing inhibition of β-amyloid toxicity to LAN-1cells by filamentous phage using the MTT assay. For the prevention assayin FIG. 5A, phages were added to 25 μM β-amyloid at molar ratios of1760:1, βA/phage. The mixtures were added to the cells. The viabilitypercentages are related to cell viability in the absence of β-amyloid(treatment-PBS), which is considered to be 100%. In FIGS. 5B and 5C,phage ability to protect against βA toxicity was measured using twopreaggregated peptides: βA1-40 and βA17-42. Both peptides (βA 1-40 andβA 17-42) were incubated for 5 days at 37° C. On the 6^(th), dayfilamentous phage was added to the preaggregated peptides at aconcentration of 1.2 nM and further incubated for 12 hours and thenadded to the cell line which was seeded in 96 well plate the day before.The cells were left to grow for another three days and then MTT wasadded. After 3 hours, extraction buffer was added and the plate wasincubated overnight at 37° C. On the following day plate was read at 570nm wavelength.

FIGS. 6A and 6B show filamentous phage injected into one hemisphere ofhAPP transgenic mice (SWE2576, Taconic) (FIG. 6B) and PBS alone injectedinto the other hemisphere (FIG. 6A). Mice that were sacrificed 3 dayspost treatment showed 40% reduction in plaque load, compared to the PBStreated hemisphere.

FIG. 7 is a graph showing the phage distribution in BALB/c mice afterintranasal application. Comparison between phage distribution in thebrain and olfactory bulb 1, 3, and 24 hours post nasal administration toBALB/c mice showed phage presence (labeled phages in uCi/mg) even onehour after administration. Their concentration was reduced after thisone hour time point.

DETAILED DESCRIPTION OF THE INVENTION

β-Amyloid peptide (βA) is one of the two hallmarks of Alzheimer'sdisease. This peptide forms fibrillar toxic aggregates in brain tissuethat can be dissolved only by strong denaturing agents. Since theseneurotoxic properties are related to peptide aggregation forms, mucheffort has been invested in developing a therapeutic approach towardsreducing or eliminating the extent of amyloid fibrillar deposition inthe brain.

Under physiological conditions, a synthetic βA adopts an aggregated formand also shows a change from a neurite, promoting a neurotoxic effect onhippocampal neurons. Aggregation of βA has been shown to depend on pH,peptide concentration, temperature, and time of incubation.

The present inventors have surprisingly discovered that filamentousphage per se has the ability to prevent βA aggregation in vitro, as wellas to dissolve already formed aggregates.

In the laboratory of the present inventors, filamentous phages M13, f1,and fd, which are well understood at both structural and genetic levels(Greenwood et al., 1991) were used. This laboratory first showed thatfilamentous bacteriophage exhibits penetration properties to the centralnervous system while preserving both the inert properties of the vectorand the ability to carry foreign molecules (Frenkel and Solomon, 2002).

Filamentous bacteriophages are a group of structurally related viruseswhich contain a circular single-stranded DNA genome. They do not killtheir host during productive infection. The phages that infectEscherichia coli containing the F plasmids are collectively referred toas Ff bacteriophages. They do not infect mammalian cells.

The filamentous bacteriophages are flexible rods about 1 to 2 micronslong and 6 nm in diameter, with a helical shell of protein subunitssurrounding a DNA core. The two main coat proteins, protein pIII and themajor coat protein pVIII, differ in the number of copies of thedisplayed protein. While pill is presented in 4-5 copies, pVIII is foundin ˜3000 copies. The approximately 50-residue major coat protein pVIIIsubunit is largely alpha-helical and the axis of the alpha-helix makes asmall angle with the axis of the virion. The protein shell can beconsidered in three sections: the outer surface, occupied by theN-terminal region of the subunit, rich in acidic residues that interactwith the surrounding solvent and give the virion a low isoelectricpoint; the interior of the shell, including a 19-residue stretch ofapolar side-chains, where protein subunits interact mainly with eachother; and the inner surface, occupied by the C-terminal region of thesubunit, rich in basic residues that interact with the DNA core. Thefact that virtually all protein side-chain interactions are betweendifferent subunits in the coat protein array, rather than withinsubunits, makes this a useful model system for studies of interactionsbetween alpha-helical subunits in a macromolecular assembly. The uniquestructure of filamentous bacteriophage enables its penetration into thebrain, although it has a mass of approximately 16.3MD and may contributeto its ability to interfere with βA fibrillization since the phagestructure resemble an amyloid fibril itself.

Considering the above, the present inventors have examined the abilityof filamentous phage to interfere with the aggregation process ofβ-amyloid peptide and found that in vitro incubation of wild-typefilamentous phage with β-amyloid peptide at different time intervals,with differing ratios, leads to prevention and/or disaggregation ofβ-amyloid. Moreover, the filamentous phage shows a protective effect oncell viability.

The most exciting data were obtained after incubation of the filamentousphage—β-amyloid fibrils with microglia cells grown on slides. Ifβ-amyloid does activate microglia, the phage dissolves it withoutactivating microglia. Phage technology provides a new and practicallyunlimited source of the anti-aggregating agent of β-amyloid, preventingthe harmful effect of antibodies which might overactivate microglia viaFc receptors.

Bacteriophages have distinct advantages over animal viruses as geneand/or delivery vehicles. They are simple systems whose large-scaleproduction and purification is very efficient and much cheaper than thatof animal viral vectors. In addition, large segments of DNA can beefficiently packaged in phagemid vectors. Having evolved for prokaryoticinfection, assembly and replication, bacteriophage can neither replicatein, nor show natural tropism for, mammalian cells. This minimizes thechances of non-specific gene delivery. Phage vectors are potentiallymuch safer than viruses as they are less likely to generate areplication-competent entity in animal cells (Monaci et al., 2001).

The present invention provides a method for inhibiting or treating braininflammation associated with amyloid deposits or involving activatedmicroglia. In addition, the present method further inhibits or treatsinflammation associated with amyloid deposits elsewhere in the bodybesides the brain, such as in the case of multiple myeloma and renalamyloidosis.

The present method involves introducing/administering to a patient inneed thereof an effective amount of a wild-type filamentousbacteriophage or a filamentous bacteriophage which does not display anantibody or a non-filamentous bacteriophage antigen on its surface. Thefilamentous bacteriophage can be any filamentous bacteriophage such asM13, f1, or fd. Although M13 was used in the Example hereinbelow, anyother filamentous bacteriophage is expected to behave and function in asimilar manner as they have similar structure and as their genomes havegreater than 95% genome identity.

When the method is used to inhibit or treat brain inflammation, thefilamentous bacteriophage is preferably administered intranasally tointroduce the active ingredient into the body of the recipient throughan olfactory system of the recipient.

The present method not only inhibits the aggregation of protein intoamyloid plaques or deposits in a plaque-forming disease, but also iseffective in disaggregating pre-formed amyloid deposits such as βAfibrils.

The anti-aggregating or disaggregating property of the filamentousbacteriophage with respect to βA fibril formation or disaggregation canbe measured by the well-known Thioflavin T (ThT) binding assay.Disrupted formation of βA fibril structure and disaggregation ofpreformed βA fibrils are indicated by a substantial decrease in ThTfluorescence.

For purposes of this specification and the accompanying claims, theterms “patient”, “subject” and “recipient” are used interchangeably.They include humans and other mammals which are the object of eitherprophylactic, experimental, or therapeutic treatment.

For purposes of this specification and the accompanying claims, theterms “beta amyloid peptide” is synonymous with “β-amyloid peptide”,“βAP”, “βA”, and “Aβ”. All of these terms refer to a plaque formingpeptide derived from amyloid precursor protein.

As used herein, “PrP protein”, “PrP”, “prion”, refer to polypeptideswhich are capable under appropriate conditions, of inducing theformation of aggregates responsible for plaque forming diseases. Forexample, normal cellular prion protein (PrPC) is converted under suchconditions into the corresponding scrapie isoform (PrPSc) which isresponsible for plaque forming diseases such as, but not limited to,bovine spongiform encephalopathy (BSE), or mad cow disease, felinespongiform encephalopathy of cats, kuru, Creutzfeldt-Jakob Disease(CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familialinsomnia (FFI).

As used herein, the term “disaggregating” refers to solubilization ofaggregated proteins typically held together by non-covalent bonds.

The term “treating” is intended to mean substantially inhibiting,slowing or reversing the progression of a disease, substantiallyameliorating clinical symptoms of a disease or substantially preventingthe appearance of clinical symptoms of a disease.

Also as used herein, the term “plaque forming disease” refers todiseases characterized by formation of plaques by an aggregating protein(plaque forming peptide), such as, but not limited to, beta-amyloid,serum amyloid A, cystatin C, IgG kappa light chain or prion protein, indiseases such as, but not limited to, early onset Alzheimer's disease,late onset Alzheimer's disease, presymptomatic Alzheimer's disease, SAAamyloidosis, hereditary Icelandic syndrome, senility, multiple myeloma,and to prion diseases that are known to affect humans, such as forexample, kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), and fatal familialinsomnia (FFI) and animals, such as, for example, scrapie and bovinespongiform encephalitis (BSE).

Because most of the amyloid plaques (also known as amyloid deposits)associated with the diseases described hereinabove are located withinthe brain, any proposed treatment modality must demonstrate an abilityto cross the blood brain barrier (BBB) as well as an ability to dissolveamyloid plaques. Normally, the average size of molecules capable ofpenetrating the BBB is approximately 2 kDa.

An increasing body of evidence shows that olfactory deficits anddegenerative changes in the central olfactory pathways are affectedearly in the clinical course of AD. Moreover, the anatomic patternsinvolved in AD suggest that the olfactory pathway may be the initialstage in the development of AD.

Olfactory receptor neurons are bipolar cells that reside in theepithelial lining of the nasal cavity. Their axons traverse thecribriform plate and project to the first synapse of the olfactorypathway in the olfactory bulb of the brain. This configuration makesthem a highway by which viruses or other transported substances may gainaccess to the CNS across the BBB.

As previously shown, intranasal administration (Mathison et al, 1998;Chou et al, 1997; Draghia et al, 1995) enables the direct entry ofviruses and macromolecules into the cerebrospinal fluid (CSF) or CNS.

Use of olfactory receptor neurons as a point of delivery for anadenovirus vector to the brain is reported in the literature. Thismethod reportedly causes expression of a reporter gene in the brain for12 days without apparent toxicity (Draghia et al, 1995).

Thus, according to a preferred embodiment of the present invention, thefilamentous bacteriophage capable of disaggregating or preventing theformation of a polypeptide aggregate associated with a plaque formingdisease or capable of inhibiting activation of microglia is deliveredvia this route to the brain.

As Aβ is produced continuously by cells in peripheral tissues whichcross the blood brain barrier (BBB) leading to localized toxic effectsin specific neuronal populations, intranasal administration of such avehicle may also prevent the progression of the disease by minimizingthe amount of peripheral Aβ available to form plagues.

A pharmaceutical preparation according to the present inventionincludes, as an active ingredient, a wild-type filamentous bacteriophageor a filamentous bacteriophage which does not display an antibody or anon-filamentous bacteriophage antigen on its surface. The pharmaceuticalpreparation can also be a mixture of different filamentousbacteriophages.

The preparation according to the present invention can be administeredto an organism per se, or in a pharmaceutical composition where it ismixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients, The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternatively, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto the brain of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired circulating antibody concentrationor titer. Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl et al, in “The Pharmacological Basis ofTherapeutics”, Ch. 1 p. 1 (1975)).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the filamentous bacteriophage which aresufficient to prevent aggregation or to disaggregate existing aggregates(minimal effective concentration, MEC). The MEC will vary for eachpreparation, but can be estimated from in vitro data. Dosages necessaryto achieve the MEC will depend on individual characteristics and routeof administration. Binding assays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration and is not intended to be limiting ofthe present invention.

Example Materials and Methods Phage Growth and Purification:

Filamentous phages (M13) were prepared from transformed TG1 cultures in2YT broth, containing 50 μg/ml kanamycin. Bacterial cells were pelletedby centrifugation, and the clarified supernatant was decanted. Phageswere precipitated from the supernatant by adding 20% of the originalvolume of 20% polyethylene glycol (molecular mass 8000 Da) in 2.5M NaCland standing at 4° C. for 3 hours. Phages were pelleted bycentrifugation (9,000 rpm, 1 hour) then resuspended in 3% of thesupernatant volume of PBS. Bacterial residues were removed by furthercentrifugation at 7000 rpm, and the phages were concentrated again byadditional polyethylene glycol precipitation. The pellet was finallyresuspended in PBS in 0.001 of the original volume of growth media.(Hart et al, 1994).

Phage Interference with β-Amyloid Aggregation:

The ability of the phage to prevent βA aggregation and to disaggregatealready formed aggregates was analyzed by three approaches:

1. Thioflavin-T Fluorescence assay:

βA aggregation was measured using Thioflavin-T (ThT) dye which changesits emission spectra upon binding to amyloid fibrils (LeVine, 1993). Toevaluate phage ability to prevent aggregates formation, 8 μl of βA1-40577 μM (Bachem), was incubated at 37° C. for 14 days, either alone(diluted to 80 μl with PBS to a final concentration of 58 μM) or withfilamentous phage (72 μl of 3×10¹² phages/ml). The fluorescence of eachreaction mixture (20 μl) was measured after addition of 980 μl ThT (2μM) (Sigma) in 50 mM glycine buffer pH9. Fluorescence was measured usinga Perkin-Elmer model LS-50 spectrofluorimeter at an excitationwavelength of 435 nm and an emission wavelength at 485 nm.

Disaggregating activity of the phage was examined as follows: βA (58 μM)was incubated for 21 days at 37° C. to promote aggregation. Phages wereadded (equal volume of phage of concentration 10¹⁴ phages/ml solution)and incubated with the aggregated βA for another 17 hours. The extent ofaggregation was evaluated using ThT fluorescence, as described above.

2. Electron Microscopy (EM):

Interaction of β-amyloid and filamentous phage. Level of βA aggregationwas visualized using EM. For the prevention of βA formation, 10 μl ofthe peptide (289 μM) was incubated either alone (20 μl PBS were added)or in combination with different phage concentrations (20 μl of phagefrom the following concentrations: 1×10¹⁴ phages/ml, 1×10¹² phages/ml,and 1×10¹⁰ phages/ml) for 9 days at 37° C.

Disaggregating activity of the phage was demonstrated by addingdifferent phage concentrations to preaggregated βA. 20 μl of β-amyloid1-40 (193 μM) dissolved in PBS were incubated at 37° C. for 10 days. Onthe 10^(th) day, 10 μl of PBS or different phage concentrations (1×10¹⁴phages/ml, 1×10¹² phages/ml) were added to the sample and incubated foran additional 16 hours.

In both the prevention and disaggregation assays, phages and βA wereadhered to carbon evaporated coated formvar 200# grids. Immunolabelingof the amyloid and phages was performed using various sizes ofgold-conjugated antibodies (Lin et al., 1999) to distinguish easilybetween β-amyloid fibrils and phage which may resemble amyloidfilaments. β-amyloid fibrils were stained with monoclonal antibody (mAb)10D5, and 2^(nd) antibody goat anti-mouse conjugated to 12 nm goldparticles (Electron Microscopy Sciences, Washington, USA). To label thephage, rabbit polyclonal sera against phage was used, and the 2^(nd)antibody was conjugated to 6 nm gold particles (Electron MicroscopySciences). The samples were negatively stained with aqueous (2% wt/vol)uranyl acetate (Sigma). The grids were visualized using JEOL 1200 EXelectron microscope at 30K and 100K magnifications.

Interaction of β-amyloid and chloroform treated phages. 200 μl of phages(10¹⁴ phages/ml) were added to 200 μl chloroform. The solution wasvortexed 6 times (10 seconds each) over 3 minute at room temperature(Griffith et al, 1981) The tube was centrifuged at 13,200 rpm for 1minute; the aqueous phase was transferred to another tube, and left inthe hood to evaporate chloroform residues. The interaction betweenβ-amyloid and spherical phages was analyzed by electron microscope.

β-amyloid (97 μM) was incubated at 37° C. for 13 days. On the 13^(th)day, PBS or different concentrations of S-phages (chloroform treated)(5×10¹³ phages/ml, 5×10¹¹ phages/ml) were added to the samples and werefurther incubated for 16 hours. The degree of β-amyloid aggregation wasvisualized by JEOL 1200 EX electron microscope as was previouslydescribed.

3. MTT Assay:

MTT assay was performed to evaluate phage protection from A toxicity onhuman neuronal cell-line viability. The assay is based on the reductionof 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)to MTT-formazan in living cells. MTT-formazan concentration was measuredby spectrophotometer at 570 nm and is directly correlated to cellviability.

LAN-1 human neuronal cell-line was cultured in RPMI supplemented with10% fetal calf serum, and 100 units/ml penicillin/streptomycin andincubated at 37° C. under 5% CO₂. For the neurotoxicity assay, culturedLAN-1 cells were seeded into a 96-well plate at a density of10⁴cells/100 μl/well. The dose-dependent neurotoxicity was measuredusing samples of β-amyloid (289 μM) either alone or with different phageconcentrations.

2 μL of the peptide were incubated with 8 μl of different phageconcentrations (5×10¹³/ml, 5×10¹²/ml, 5×10¹¹/ml phages) for 4 days at37° C. in order to examine phage preventive effect against βAneurotoxicity. The samples were added 24 hours after the cells wereseeded and attached to the plate. The plates were incubated at 37° C.for 2 days, after which cell viability was assessed by measuringcellular redox activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as was previously described (Solomon et al,1997). MTT (Sigma) was added to the wells at a final concentration of 1mg/ml and incubated with the cells for a further 3 hours at 37° C. Celllysis buffer (20% wt/vol SDS in a solution of 50% dimethylformamide, pH7.4) was added, and the plate was incubated overnight at 37° C. MTTreduction was determined colorimetrically by the change in OD at 570 nmusing an automated microplate spectrophotometer.

In the disaggregation assay of already formed βA fibrils, two peptideswere used (β-amyloid 17-42 and β-amyloid 1-40) as the neurotoxicfactors. The ability of the phage to protect the cell viability from theaggregated peptides was examined as follows below. In the firstexperiment, the cells were incubated for 5 days with preaggregated βA1-40, while in the second experiment, β-amyloid 17-42 was used as theneurotoxic peptide. As was previously described, the cells were seededinto a 96-well plate at a density of 10⁴cells/100 μl/well and allowed toattach for 24 hours before the samples were added. To 2 μl of theaggregated peptide (288 μM-stock solution), 8 μl of the phages (10¹⁴phages/ml, 10¹³ phages/ml, 10¹² phages/ml) were added and incubatedovernight. The samples were added to the cells, and were furtherincubated for two days. MTT at a final concentration of 1 mg/ml wasadded, and lysis buffer was put 3 hours later to enable measurement ofMTT-formazan content. Cell viability was assessed by quantifying theO.D. change in 570 nm.

In-Vivo Application of Filamentous Phage:

Wild-type, as well as transgenic mice, model of AD were challenged witha filamentous phage:

Evaluation of anti-aggregating properties. In order to assess maximumpotential of phage activity as an anti-aggregating agent, phages wereinjected intracranially to transgenic mice (Taconic, APPSWE(2576), 10month-old) as follows:

2.5 μl the filamentous phage solution (10¹⁴ phages/ml) was injected over10 minutes (Bregma −2.8 mm, lateral 2.5 mm, ventral 2.5 mm) to onehemisphere, while to the contra-lateral side, phosphate-buffer-saline(PBS) was applied as a control (FIG. 1). Since the time period requiredfor phages to disaggregate amyloid deposits is unknown, the treated micewere sacrificed at different time points. The first group wasintracardially perfused using 4% paraformaldehyde one hourpost-injection, the second group—two days after administration, and thelast group—after 3 days. Their brains were post-fixed overnight in 4%paraformaldehyde, and cut using a microtome. Thioflavin-S (ThS) stainingwas performed to evaluate plaque load. For this purpose, the sectionswere stained with Mayer's hematoxylin to quench nuclear autofluorescenceand after washing, ThS solution (1%) was applied for 3 minutes.Differentiation was done using 1% acetic acid for 20 min, and afterwashes the slides were dried and mounted with anti fade mounting medium.Plaque load was calculated using LEICA Qwin program.

Biodistribution of radioactive labeling of filamentous phage.Filamentous phages were radioactively labeled with I¹²⁵. The phages werepurified from the unbound iodine using G25 sephadex column and theeluate was further precipitated using polyethylene-glycol (PEG) as waspreviously described. 9 BALB/c mice were divided into 3 groups. Eachmouse received intranasally 100 μl of phages (1.25×10¹² phages) over anhour, labeled with 9 microcurie I¹²⁵. The first group of mice wassacrificed an hour after administration of intra-cardial perfusion using4% paraformaldehyde. The second group was sacrificed 3 hourspost-treatment, and the last group, after 24 hours. After perfusion, thebrains as well as the periphery organs were removed and their gammaradiation was measured.

Intranasal administration of filamentous phage. To fully evaluate theeffect of filamentous phage, the phages were administered intranasallyto SWE/APP2576 transgenic mice (Taconic, 10 month-old). 20 μl of phagesolution (5×10¹²/ml) were applied every two weeks, for 4 months andcognitive functions were evaluated. After the ninth immunization, anobject recognition test was carried out to study the influence of phageon memory improvement. On the first day, mice were exposed to two newobjects for 20 minutes. On the following day, one object was replaced,and the curiosity of the mice to explore the novel item was tested.Recognition index was calculated for each mouse by dividing the time itspent near the new object by the total time it spent near both objects.Thus, values above 0.5 are indicative for recognizing the old item andspending more time around the new object for its investigation.

Results

In vitro anti-aggregating properties of filamentous phages wereevaluated regarding reduction and prevention of amyloid formation. Inthe Thioflavine-T assay, the phages were more efficient indisaggregating βA fibrils than in preventing their formation. A declineof 26% in βA aggregation was observed (FIG. 2B) when the peptide wasincubated with filamentous phage in a molar ratio 1:10,000 (phage toβA). Phage addition (28 nM) to preaggregated βA resulted in 45%reduction in amyloid fibrils (FIG. 2A).

Visualization of the physical interaction between phages and β-amyloidpeptide by electron microscopy was made using immunogold-stainedβ-amyloid and its complexes with phages. When phages interact withβ-amyloid peptide, the aggregates are smaller and more diffused comparedto phage absence. Interestingly, incubation of phages with β-amyloidpeptide led to phage degradation, as can be learned from the massivelabeling of antibody against phage major coat protein not on phageparticles (FIG. 3C). This phenomenon was not observed when the phageswere incubated with β-amyloid peptide overnight (in the disaggregationassay) or when phages were incubated alone for the same period of time(FIG. 3D). FIG. 3(F-K) shows the effect of phages on formed amyloidaggregates. FIG. 3F demonstrates amyloid fibrils in a cluster labeled bythe 12 nm gold particle. Higher magnification (FIG. 3G) shows thedifferent sizes of aggregated fibril needles. FIGS. 3H-K present phagesorganized in bundles (arrow head) which are dispersed when attached tothe amyloid needles (arrow). The amyloid cluster is small and containssmall narrow amyloid fibrils. Of special interest is the parallelorganization of the phages to the amyloid fibrils (open arrow).

The pVIII molecule has been shown to exist in three conformations. Inthe intact phage, the protein is at least 90% α-helical, but largechanges in shape occur upon exposure to chloroform/water interface. Thisis a temperature-dependent process. Rods (I-forms) are formed at 2° C.while spherical structures (S-forms) are formed at 25° C. The conversionto S-form occurs with a substantial decrease in the helix content of thecoat protein but with a significant change in the environment oftryptophan 26 (Roberts and Dunker, 1993).

Treatment of the phages with chloroform for three minutes showed thatalthough spherical phages are present at the site of βA aggregates, theyare usually located at the end of the fibrils and do not contribute tosolubilization (FIGS. 4A-B).

MTT:

Filamentous phage protection on LAN-1 cells from βA neurotoxicity wasdemonstrated using the MTT assay. When phages were incubated with βA,neuronal viability increased compared to cells that grew in the presenceof βA alone. The highest amount of phages that was added to the cellculture caused the most significant effect (17% increase in cellviability) (FIG. 5A).

Phage protection from β-amyloid 17-42 toxicity was higher compared toits ability to prevent β-amyloid 1-40 toxicity. When phages were addedto the preaggregated βA 17-42, cell viability was increased by 30%,compared to cells that grew with βA alone (FIG. 5B). Addition of thesame phage concentration to βA 1-40 resulted in only 17% increase incell viability (FIG. 5C). Addition of higher concentration of phagessucceeded to increase cell viability to 25% compared to βA treatedcells.

A possible reason for this difference could be attributed to theN-terminus of β-amyloid that may interfere with phage activity, or dueto the fact that β-amyloid 17-42 aggregates faster than β-amyloid 1-40(Pike et al., 1995). Phage affinity to β-amyloid 1-40 increases with thepeptide aggregation. ELISA, which checked phage binding to solubleβ-amyloid, 1 hour aggregated β-amyloid or 3 weeks aggregated peptide,showed significant difference in phage binding to βA 1-40. Therefore thehigher affinity of phages to the highly aggregated peptide may explainits stronger interactions with β-amyloid 17-42.

In the in-vivo studies of disaggregation of β-amyloid plaques byintracerebral injection of filamentous phages, no reduction in plaqueload was detected in mice sacrificed one hour post injection. In micesacrificed two days after treatment, 25% reduction in plague load wasnoticed in the hemisphere treated with filamentous phage compared to PBSinjected hemisphere. Mice sacrificed three days after treatment showed40% reduction in plaque load in the phage-injected hemisphere (FIGS. 6Aand 6B).

Brain Distribution of Radioactive Labeled Filamentous Phages:

Following intranasal administration, filamentous phages were detected inthe olfactory bulb and in the brain as early as 1 hour postadministration (FIG. 7). Although the groups were too small (n=3) toconclude phage clearance rate, it seems that phage concentration reachesits peak after an hour, and is then eliminated almost completely within24 hours. The high variability between the mice can be the result ofswallowing and inhaling different amounts of phages. The penetrated doseto brain parenchyma varied from 0.0009% to 0.018% of the given dose. Itis worth mentioning that 0.1% of antibody sera concentration succeededin reaching the brain and reduce plaque load. These preliminary datashow that phage can penetrate the brain in a short time and startclearance after an hour in normal BALB/C mice. Therefore, intranasaladministration of filamentous phage can be tested for its efficiency intreating transgenic mice bearing amyloid deposits.

Repeated Intranasal Administration of Filamentous Phage into TransgenicMice:

Significant improvement in cognitive functions was noticed in thephage-treated mice compared to the non-treated group (data not shown).At the end of the study, histochemical analysis will be performed toevaluate mice plaque load, microglia activation, synapses density andprobable adverse effects in order to get a comprehensive estimation oftreatment efficiency.

Discussion

The laboratory of the present inventors have previously reported thatsite-directed monoclonal antibodies (mAbs) towards the N-terminal regionof βa, bind preformed βA fibrils, and lead to amyloid disaggregationinto an amorphous state and inhibit their neurotoxic effect (Solomon etal., 1996, 1997). This activity was also demonstrated in vivo intransgenic mice for AD (Bard et al., 2000). Phage displaying scFvagainst βA to deliver intranasally anti-aggregating antibody fragment tobrain plaques was used (Frenkel et al., 2002). The phage was used as avehicle which enabled scFv penetration to the brain and its binding toamyloid deposits in transgenic mice.

A direct correlation was shown between the number of applications andthe amount of phage detected in the hippocampus and olfactory bulb. Thelinear structure of the phage is suggested to confer penetrationproperties via various membranes. It is worth mentioning that nopathological signs were observed in those mice brains after phageadministration (Frenkel et al., 2002).

βA fibrils were visualized both by ThT and fluorescent labeledanti-phage antibodies and the disappearance of filamentous phage fromthe brain without inducing toxic effect was shown in histology studies.In previously reported experiments, filamentous phages wereintravenously injected into mice and were subsequently rescued from thedifference organs, showing that their integrity was not affected duringcirculation in the body.

Amyloid fibrillization is considered to be driven by hydrophobic ratherthan electrostatic interactions. βA contains two hydrophobic segments,the central part of which is composed of residues 17-21 and theC-terminal region that contains residues 30-40. According to the modelbased on experimental constraints from solid state NMR, the peptideconformation contains two β-strands, separated by a 180° bend formed byresidues 25-29. The β-strands form two in-register parallel β-sheets,which interact through sidechain-sidechain contacts. The hydrophobicsidechains are exposed to the outer surface and form a hydrophobic face,while the other charged and polar sidechains are distributed on theopposite face, on the convex side of the bend, and in the N-terminalsegment where they could be solvated as the fibrils grow (Petkova et al,2002).

On the other hand, the major phage coat protein is composed from threesections: the outer surface, occupied by the N-terminal region of thesubunit, rich in acidic residues that interact with the surroundingsolvent and give the virion a low isoelectric point; the interior of theshell, including a 19-residue stretch of apolar side-chains, whereprotein subunits interact mainly with each other; and the inner surface,occupied by the C-terminal region of the subunit, rich in basic residuesthat interact with the DNA core. The fact that virtually all proteinside-chain interactions are between different subunits in the coatprotein array, rather than within subunits, makes this a useful modelsystem for studies of the interactions between α-helix subunits in amacromolecular assembly.

In the present study, it was demonstrated that phages possessanti-aggregating properties in addition to its reported function as avehicle for delivering antibodies against βA to the brain. From the datapresented in the Example, filamentous phage interacts with β-amyloid andcan interfere with its aggregation process and even induce itssolubilization. Disaggregating property is of great value since, atpresent, diagnosis is made in late stages of the disease when β-amyloidplaques are already formed. This process is time dependent since afterintracranial injection of phages into Tg mice bearing amyloid plaques,maximum effect was observed only after 3 days. This effect can be theresult of the phage's unique structure as a long thin filament whichenables it to organize along the amyloid fibrils, as can be seen in theelectron microscopy micrographs. This theory is evidenced by the factthat spherical phages which lost their linear structure could notinhibit amyloid formation. Another factor that can be a majorcontributor to phage activity is its high content of alpha-helices(protein 8) which may interfere with beta sheet structure. The pVIIIsubunits pack in a helical array to form a tubular structure whichsurrounds the ssDNA genome. The C-terminal end of pVIII is exposedtowards the inside of the tubular structure and contains positivelycharged residues which interact with the negatively charged ssDNAgenome. The middle portion is rich in hydrophobic amino acids which areresponsible for the interaction among subunits. Finally, the flexible,negatively charged N-terminal portion is exposed to the outside of theparticle (Marvin, 1998).

Filamentous bacteriophages offer an obvious advantage over othervectors. The filamentous phages, M13, f1, and fd, are well understood atboth structural and genetic levels (Wilson and Finley 1998; Rodi andMakowski, 1999). They were genetically engineered to display bothantigen and/or antibody and were used in different biological systems topresent foreign proteins on their surfaces (Scott et al. 1990;McCafferty et al 1990). Having evolved for prokaryotic infection,assembly and replication, bacteriophage can neither replicate in, norshow natural tropism for mammalian cells. This minimizes the chances ofnon-specific gene delivery. Phage vectors are potentially much saferthan other viruses, as they are less likely to generate areplication-competent entity in animal cells.

Another benefit in using bacteriophage as disaggregating agent is itseasy production, since more material can be easily obtained by growth ofbacterial cultures. According to the radioactive labeling assay, phagepenetrate to brain parenchyma after less than an hour. Their eliminationprobably begins soon after. The percentage of the penetrated phage fromthe administered dose varied from 0.0009% to 0.018%. More studies shouldbe performed to minimize the variability between the mice. Minimizingthe dose, and waiting longer between each drop can help. In comparison,0.1% from antibody sera concentration reaches the brain. Although themolecular weight of the phage is two orders of magnitude higher thanantibody, intranasal application can result in phage penetration.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

-   Banks and Kastin, Prog Brain Res., 91:139-4 (1992)-   Bard F, Cannon C, Barbour R, Burke R L, Games D, Grajeda H, Guido T,    Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M,    Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K,    Welch B, Seubert P, Schenk D, Yednock T. Peripherally administered    antibodies against amyloid beta-peptide enter the central nervous    system and reduce pathology in a mouse model of Alzheimer disease.    Nat Med. August; 6(8):916-9 (2000)-   Caughey et al, “Secondary structure analysis of the    scrapie-associated protein PrP 27-30 in water by infrared    spectroscopy”, Biochemistry 30:7672-7680 (1991)-   Chartier Harlan et al, Nature 353:844 (1991)-   Chou et al, Biopharm Drug Dispos. 18(4):335-46 (1997)-   De Gioia et al, “Conformational polymorphism of the amyloidogenic    and neurotoxic peptide homologous to residues 106-126 of the prion    protein”, J Biol Chem 269:7859-7862 (1994)-   Draghia et al, Gene Therapy 2:418-423 (1995)-   Forloni et al, “Neurotoxicity of a prion protein fragment”, Nature    362:543-546 (1993)-   Frenkel and Solomon, PNAS, 99:5675-5679 (2002)-   Frenkel D, Solomon B. Filamentous phage as vector-mediated antibody    delivery to the brain. Proc Natl Acad Sci USA. April 16;    99(8):5675-9 (2002)-   Frenkel et al, “N-terminal EFRH sequence of Alzheimer's β-amyloid    peptide represents the epitope of its anti-aggregating antibodies”,    J Neuroimmunology 88:85-90 (1998)-   Gajdusek, Science 197:943-960 (1991)-   Gazit E., Mechanistic studies of the process of amyloid fibrils    formation by the use of peptide fragments and analogues:    implications for the design of fibrillization inhibitors. Curr Med    Chem. October; 9(19):1725-35 2002-   Goate et al, Nature 349:704 (1991)-   Greenwood et al., J. Mol. Biol., 220:821-827 (1991)-   Griffith J., Manning N., Dunn K, Filamentous bacteriophage contract    into hollow spherical particles upon exposure to a chloroform-water    interface. Cell. March; 23(3):747-53 (1981)-   Hardy, TINS, 20:154 (1997)-   Hart S L, Knight A M, Harbottle R P, Mistry A, Hunger H D, Cutler D    F, Williamson R, Coutelle C. Cell binding and internalization by    filamentous phage displaying a cyclic Arg-Gly-Asp-containing    peptide. J Biol Chem. April 29; 269(17):12468-74 (1994)-   Horiuchi and Caughey, “Specific binding of normal prion protein to    the scrapie form via a localized domain initiates its conversion to    the protease-resistant state”, EMBO J. 18:3193-3203 (1999)-   Kanyo et al, “Antibody binding defines a structure for an epitope    that participates in the PrPC—>PrPSc conformational change”, J Mol.    Biol. 293:855-863 (1999)-   LeVine H 3^(rd), Thioflavine T interaction with synthetic    Alzheimer's disease beta-amyloid peptides: detection of amyloid    aggregation in solution. Protein Sci. March; 2(3):404-10 (1993)-   Lin J. Yanase K, Rutgers A, Madaio M P, Meyers K B, Selection of    specific phage from display libraries: monoclonal antibody against    VCS M13 helper phage coat protein III (gIIIp). Hybridoma, June;    18(3):257-61 (1999)-   Maggio J E, Mantyh P W, Brain amyloid—a physicochemical perspective.    Brain Pathol. April; 6(2):147-62 (1996)-   Marvin D A, Hale R D, Nave C, Citterich M H, Molecular models and    structural comparisons of native and mutant class I filamentous    bacteriophages Ff (fd, f1, M13), If1 and Ike. J. Mol. Biol.    235:260-286 (1994)-   Marvin D A. Filamentous phage structure, infection and assembly.    Curr Opin Struct Biol. April; 8(2):150-8 (1998)-   Mathison at al, J. Drug Target, 5(6):415-441 (1998)-   McCafferty J, Griffiths A D, Winter G, Chiswell D J. Phage    antibodies: filamentous phage displaying antibody variable domains.    Nature. December 6; 348(6301):552-4 (1990)-   Medori, Tritschler et al., N Engl J Med 326: 444-449 (1992)-   Monaci P., et al., Curr Opin Mol Ther., 3(2):159-69 (2001)-   Mullan et al, Nature Genet. 1:345 (1992)-   Murrell et al, Science 254:97 (1991)-   Pan et al, “Conversion of alpha-helices into beta-sheets features in    the formation of the scrapie prion proteins”, Proc Natl Aced Sci USA    90:10962-10966 (1993)-   Peretz et al, “A conformational transition at the N terminus of the    prion protein features in formation of the scrapie isoform”, J Mol    Biol 273:614-622 (1997)-   Pike C J, Overman M. J, Cotman C. W, Amino-terminal deletions    enhance aggregation of beta-amyloid peptides in vitro. J Biol. Chem.    October 13; 270(41):23895-8 (1995)-   Roberts L M, Dunker A K, Structural changes accompanying    chloroform-induced contraction of the filamentous phage fd.    Biochemistry. October 5; 32(39):10479-88 (1993)-   Rodi, D J. and Makowski, L., Phage-display technology—finding a    needle in a vast molecular haystack. Curr. Opin. Biotechnol.,    10:87-93 (1999)-   Scott, J K.; and Smith, G P, Searching for peptide ligands with an    epitope library. Science, 249: 386-390 (1990)-   Selvaggini et al, “Molecular characteristics of a    protease-resistant, amyloidogenic and neurotoxic peptide homologous    to residues 106-126 of the prion protein”, Biochem Biophys Res    Commun 194:1380-1386 (1993)-   Silen and Agard, “The alpha-lytic protease pro-region does not    require a physical linkage to activate the protease domain in vivo”,    Nature 341:462-464 (1989)-   Solomon B, Koppel R, Frankel D, Hanan-Aharon E., Disaggregation of    Alzheimer beta-amyloid by site-directed mAb. Proc Natl Acad Sci USA.    April 15; 94(8):4109-12 (1997)-   Solomon B, Koppel R, Hanan E, Katzav T., Monoclonal antibodies    inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid    peptide. Proc Natl Acad Sci USA. January 9; 93(1):452-5 (1996)-   Tagliavini et al, “Synthetic peptides homologous to prion protein    residues 106-147 form amyloid-like fibrils in vitro”, Proc Natl Acad    Sci USA 90:9678-9682 (1993)-   Wilesmith and Wells, Curr Top Microbiol Immunol 172:21-38 (1991)-   Wilson, D R. and Finlay, B B., Phage display: applications,    innovations, and issues in phage and host biology. Can. J.    Microbiol. 44: 313-329 (1998)-   Young A A. et al, FEES Lett, 343(3):237-41 (1994)

1. A pharmaceutical composition comprising a non-radioactively labelledwild-type filamentous bacteriophage or a non-radioactively labelledfilamentous bacteriophage which does not display an antibody or anon-filamentous bacteriophage antigen on its surface; and apharmaceutically acceptable carrier or excipient.
 2. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition is inunit dosage form.
 3. The pharmaceutical composition of claim 2, whereinthe filamentous bacteriophage is selected from the group consisting ofM13, f1, fd, and mixtures thereof.
 4. The pharmaceutical composition ofclaim 3, wherein the filamentous bacteriophage is a wild-type M13bacteriophage.
 5. The pharmaceutical composition of claim 1, wherein thefilamentous bacteriophage is selected from the group consisting of M13,f1, fd, and mixtures thereof.
 6. The pharmaceutical composition of claim5, wherein the filamentous bacteriophage is a wild-type M13bacteriophage.
 7. A method for reducing the amount of amyloid plaque ina subject suffering from a plaque forming disease, comprisingadministering to the subject in need thereof an amount of either awild-type filamentous bacteriophage or a filamentous bacteriophage whichdoes not display an antibody or a non-filamentous bacteriophage antigenon its surface effective to reduce the amount of amyloid plaque in thesubject and by a route of administration that causes the bacteriophageto come into contact with the amyloid plaque.
 8. The method of claim 7,wherein the plaque forming disease is selected from early onsetAlzheimer's disease, late onset Alzheimer's disease, presymptomaticAlzheimer's disease, SAA amyloidosis, hereditary Icelandic syndrome,senility, multiple myeloma, kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia(FFI), scrapie, bovine spongiform encephalitis (BSE), and other diseasescharacterized by formation of plaques by an aggregating protein selectedfrom beta-amyloid, serum amyloid A, cystatin C, IgG kappa light chainand prion protein.
 9. The method of claim 8, wherein the plaque formingdisease is selected from early onset Alzheimer's disease, late onsetAlzheimer's disease, presymptomatic Alzheimer's disease, kuru,Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease(GSS), fatal familial insomnia (FFI), scrapie, bovine spongiformencephalitis (BSE), and other diseases characterized by formation ofplaques by an aggregating protein selected from beta-amyloid and prionprotein.
 10. The method of claim 9, wherein the plaque forming diseaseis selected from early onset Alzheimer's disease, late onset Alzheimer'sdisease and presymptomatic Alzheimer's disease.
 11. The method of claim8, wherein the filamentous bacteriophage is administered by a route ofadministration that causes the bacteriophage to come into contact withamyloid plaque present in the brain of the subject.
 12. The method ofclaim 11, wherein the route of administration is selected fromintrathecal and direct intraventricular.
 13. The method to claim 12,wherein the administration is by bolus injection or continuous infusion.